Some comments and analysis from the exciting and fast moving world of Genomics. This blog focuses on next-generation sequencing and microarray technologies, although it is likely to go off on tangents from time-to-time

Pages

Thursday, 10 October 2013

Hack your MiSeq and get $400 off a 600bp run

I’ll start off by saying not quite, but you can read on to get an idea on how to increase read length of a MiSeq 500cycle v2 kit to get 600bp of data.

MadsAlbertsen posted on a SEQanswers thread about their protocol to squeeze a little more out of the MiSeq. They are using a hacked MiSeq Reagent kit v2 (500 cycles) and running a 2x301bp which is not supported by Illumina. “Do it at your own risk! (although it works nicely.)” is the message on the website. The group are using the modified protocol and hacked kits for bacterial 16S rRNA gene amplicon sequencing of the V1-3 variable region. The target region in E. coli position is a total of 489bp, but depending on target species can vary up to significantly (making the 2x301 run necessary).

How to hack your MiSeq kit: Make sure you follow the instructions on adding a little extra reagent to some of the wells. 5 mL of incorporation buffer from well 1 of a left-over reagent cartridge to well 1 of the 2x301 cartridge.7 mL of scan mix from well 2 of a left-over reagent cartridge to well 2 of the 2x301 cartridge.6.8 mL of cleavage mix from well 4 of a left-over reagent cartridge to well 4 of the 2x301 cartridge.80 mL of incorporation buffer from a left-over incorporation buffer bottle.

Now simply set the Miseq to 2x301 in the samplesheet and ignore the warning the software gives. Et voila 600bp for the price of 500. With a MiSeq v3 kit costing about $1400 that’s potentially a $400 saving.

Will we be doing this in my lab? No way, I’m far too conservative with users samples to play around like this. But I wish I could do more stuff like this, as it’s fun. It makes me want to come up with my own genomics Instructables.

Over the last 16 years he's worked at; the Norfolk & Norwich Hospital on ErbB2, Royal London on Diabetes genetics, the Cambridge Uni Department of Pathology on Immunology group and the John Innes Centre on Wheat disease resistance gene cloning and arrays. In 2000 he set up an Affy and spotted microarray facility at JIC, he co-founded the UK Affy user group, which is still going strong. At JIC he also won a Biotech competition, and hopes one-day to start a business. Although none of his ideas have come to anything yet!

In 2006 James moved to set up the genomics facility at CRUK's new Cambridge Institute. Today the lab offers primarily NGS services on Illumina HiSeq, MiSeq & NextSeq for scientists at CRUK-CI and nine other University departments, as well as single-cell analysis on Fuildigm C1 and 10X Genomics.